Male 21-day-old SD rats (n=30) were obtained from the Experimental Animal Center of Nantong University (Nantong, China). The animals were maintained at controlled temperature (25±2°C) and constant photoperiodic conditions (12 h light:12 h dark). The rats were given ad libitum access to food and water. The experimental procedure was designed in compliance with the Recommendations for the Care and Use of Laboratory Animals of the Local Ethical Committee at the Experimental Animals Center of Nantong University.

Unilateral cryptorchidism was induced by the surgical procedure described previously (12,20). In brief, SD rats (n=24) were anesthetized using Nembutal (40 mg/kg body weight; concentration, 1%; BOC Sciences, Shirley, NY, USA) and, following a small incision to the abdomen, the gubernaculum was cut on one side to displace the testis into the abdomen. Testis descent was prevented by closure of the inguinal canal on one side by suturing (Hualikang Medical Instrument Co., Nantong, China). Following surgery, the animals were housed and fed routinely with analgesics (Brufen, 1 mg/ml; GlaxoSmithKline, Shanghai, China). The rats were then divided into four groups, a high-dose NGF group (HN group; n=6), low-dose NGF group (LN group; n=6), human chorionic gonadotropin (HCG) group (HC group; n=6) and negative control group (NC group; n=6). In the HN and LN groups, 9,000 and 45,00 IU murine NGF (NOBEX; Xiamen Bioway Biotech, Xiamen, China) were injected intramuscularly each day for 5 days, respectively. This commenced from the second day following surgery. The HC group were administered with 400 IU HCG intramuscularly and the NC group were administered with normal saline intramuscularly. A total of six normal SD rats without surgery were used as a blank control group (B group) and were housed in the same cage.

The normal and undescended testes were removed and decapsulated 39 days following surgery at sexual maturity, which was calculated using the website, http://www.taletn.com/rats/age/. The samples were either stored at −70°C or fixed for further use. All surgical procedures were performed under ether anesthesia (2–4%; Jiangsu Qiangshen Chemical Co., Jiangsu, Chinag) and the animals were sequentially sacrificed by CO₂ asphyxiation.

Total RNA from the decapsulated testes was extracted using TRIzol reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer’s instructions. To avoid possible genomic DNA contamination, all the RNA samples were treated using RNase-free DNase (Promega Corporation, Madison, WI, USA). One step RT-qPCR was performed using a SensiMixTM One-Step kit (Quantace, London, UK) according to the manufacturer’s instructions of the Rotor-Gene 3000 real-time DNA analysis system (Corbett Research, Sydney, Australia). For RT-qPCR examination of the temporal expression of β-NGF and Hox10, the first-strand cDNA was synthesized using an Omniscript reverse transcription kit (Qiagen, Hilden, Germany) in a 20 μL reaction system containing 2 μg total RNA, 0.2 U/μL M-MLV reverse transcriptase, 0.5 mM dNTP mix and 1 μM Oligo-dT primer. The cDNA was diluted 1:5 prior to use in the RT-qPCR assays. Subsequently, the one step RT-qPCR was performed using a SensiMixTM One-Step kit (Quantace, London, UK) according to the manufacturer’s instructions of the Rotor-Gene 3000 real-time DNA analysis system (Corbett Research). The mRNA expression levels of β-NGF and Hox10 were calculated using Rotor-Gene Real-Time Analysis software 6.1 (Build 90; Corbett Research). The oligonucleotide primers of β-NGF, Hox10 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in Table I. The amplification program consisted of 30 min at 42°C for reverse transcription and 2 min at 95°C for Taq activation, followed by 45 cycles of 95°C for 15 sec, 58°C for 30 sec and elongation at 72°C for 40 sec. GAPDH was used as an endogenous control gene for normalization.

The DIG-labeled-β-NGF RNA sense and antisense probes were generated using a DIG RNA Labeling kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, the total RNA from the brain tissue of three SD rats was extracted using TRIzol reagent, as previously mentioned, and the cDNA fragment of β-NGF was obtained from the total RNA using RT-qPCR with the following primers: 5′-GATCGGCGTACAGGCAGAAT-3′ and 5′-GGCTCGGCACTTGGTCTCAA-3′. The resulting product was then recovered from agarose gel and cloned into a pGEM-T easy vector (Promega Corporation) using T4 DNA ligase (Promega Corporation). The recombinant plasmid was transformed into the Escherichia coli strain, DH5a (Biomiga, Inc., San Diego, CA USA) and positive clones, confirmed by DNA sequencing, were transformed into E. coli Top10 (Biomiga, Inc.). The successfully-transformed E. coli Top10 was collected from a single colony and was then grown overnight at 37°C in Luria-Bertan medium supplemented with ampicillin (Sigma-Aldrich, St. Louis, MO, USA). Following plasmid extraction, the plasmid was 1inearized using the restriction enzymes, Apa I and Sal. Using Sp6 and T7 RNA polymerase, respectively, the DIG-labeled sense and antisense probes were transcribed in vitro using a DIG RNA Labeling kit according to the manufacturer’s instructions. The sense probe was synthesized by T7 RNA polymerse, while the antisense probe by T3 RNA polymerse. In order to confirm the quality of the probes, an agarose gel was used, which revealed the antisense probe as a clear specific band and the sense probe also as a bright band

In situ hybridization (ISH) histochemistry was performed to detect the mRNA expression of β-NGF mRNA in the 4-μm frozen sections from the decapsulated testes, which were fixed in freshly prepared 4% paraformaldehyde (pH 7.4) and stored at 4°C for 8 h. The sections were then dried at 43°C overnight and washed twice with 0.1% diethypyrocarbonate-treated water. Following pre-hybridization for 2 h, the sections were hybridized with DIG labeled-β-NGF RNA probes (1 μg/ml) in a humid chamber at 42°C for 18 h. These sections were then washed twice with 2X saline sodium citrate (SSC)/0.1% sodium dodecyl sulfate (SDS) buffer at 25°C for 5 min and with 1X SSC/0.1% SDS at 60°C for 15 min. Following incubation in inhibiting solution (100 mM maleic acid, 150 mM NaCl and 1% blocking reagent) at 37°C for 30 min, the sections were incubated with the anti-DIG-AP (Roche Diagnostics, GmbH) at 1:5,000 dilutions in the inhibiting solution at 4°C overnight. The sections were then washed twice using the 100 mM maleic acid buffer (pH 7.5) and 0.3% solution of Tween-20 (15 min each at 25°C), followed by the addition of the detection buffer, containing 100 mM Tris-HCl (pH 9.5) and 100 mM NaCl, for 5 min. The ISH signals were detected using a coloring solution consisting of NBT/BCIP (Roche Diagnostics GmbH) in the dark for 3–12 h. The coloring reaction was terminated using 100 mM phosphate-buffered saline (PBS; Hyclone Laboratories, Inc., Logan, UT, USA) and images were captured under a BX51 Olympus microscope (Olympus, Tokyo, Japan). A control experiment was performed in parallel using a sense probe. No significant β-NGF mRNA signal (blue) was observed. Bar =100 μm.

The frozen decapsulated testicular tissues were cut into 4-μm slices. The sections were then inhibited in PBS with 5% goat serum (Gibco-BRL, Carlsbad, CA, USA) for 15 min at room temperature and incubated with goat anti-rat-β-NGF polyclonal antibody (1:400, R&D Systems, Minneapolis, MN, USA) containing 1% bovine serum albumin (BSA) at 4°C for 24 h. Following washing with PBS, these sections were incubated with monoclonal anti-fluorescein isothiocyanate-conjugated mouse anti-goat immunoglobulin G antibody (1:1,000; Sigma-Aldrich, St Louis, MO, USA) for 2 h at room temperature. The samples were then washed with PBS and the samples were observed using fluorescence microscopy (Zeiss Axioskop 40; Zeiss, Oberkochen Germany). As mentioned above, the IF experiments were repeated at least three times. Negative controls were included by replacement of the primary antibody with PBS.

The decapsulated testicular tissues were fixed in formalin and embedded in paraffin. The serial sections were cut at 4 μm on a manual rotary microtome (Leica RM2235; Leica Microsystems GmbH, Wetzlar, Germany). IHC was performed on the sections using an Autostainer Universal Staining system (LabVision, Fremont, CA, USA). The sections were deparaffinized and peroxidase was quenched using methanol and 3% H₂O₂ for 15 min. For antigen retrieval, the sections were boiled under pressure in citrate buffer (pH 6.0) for 3 min. Nonspecific binding was inhibited using 5% goat serum in PBS for 15 min and the tissues were incubated with rabbit anti-HoxA10 antibody (1 μg/ml; H-90; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) containing 1% BSA at 4°C for 24 h. Following washing with PBS, the sections were incubated with horseradish peroxidase-conjugated goat anti-rabbit antibody (DakoCytomation, Carpinteria, CA, USA) for 15 min at room temperature. To develop the colour, the sections were incubated for 15 min with diaminobenzidine solution (Kem-En-Tec Diagnostics, Taastrup, Denmark) and the sections were weakly counterstained using hematoxylin. Negative controls were included by replacement of the primary antibody with PBS.

The decapsulated testicular tissues were fixed in 1% paraformaldehyde/1.25% glutaraldehyde in 0.1 M PBS, post-fixed in 1% buffered osmium tetroxide, dehydrated through a graded ethanol series and embedded in Epon 812 epoxy resin. The 1.0 μm sections for orientation were stained using toluidine blue. The ultra-thin sections (50 nm) were stained using uranyl acetate and lead citrate, followed by examination using a transmission electron microscope (JEM-1230; JEOL Ltd., Tokyo, Japan).

The mRNA expression levels of HoxA10 and β-NGF mRNA in the testis, measured using one step RT-qPCR, were assessed using a one way analysis of variance test paired with a t-test. P<0.05 was considered to indicate a statistically significant difference and the data were analyzed using STATA 9.0 software (Stata Corporation, College Station, TX, USA).

Total RNA was extracted from the testis and subjected to one-step RT-qPCR to investigate the mRNA expression levels of HoxA10 and β-NGF in the groups with various treatments. The expression of HoxA10 and β-NGF were individually normalized to GAPDH, expressed as the ratio in the same sample at day 39 following surgery (Fig. 1A and B). Among the groups, the highest levels of mRNA expression of the two genes were observed in the HN group (HoxA10, F=125.58, P<0.001; and β-NGF, F=49.03, P<0.001). Compared with the testes in the NC group, the mRNA expression of HoxA10 and β-NGF was clearly upregulated in the HN and LN groups.

The mRNA signals of β-NGF were observed as blue staining in the cytoplasm of the germ cells in the testes. Compared with the NC group, the mRNA expression of β-NGF in the undescended testes increased on administering a high dose of exogenous NGF (Fig. 2A–E). This result was consistent with that of the above-mentioned one-step RT-qPCR.

The β-NGF protein was stained in light green using IF, which revealed that the majority was localized in the cytoplasm of the germ cells, interstitial cells and leydig cells. The β-NGF protein was expressed in all the testicular tissues in each group, including the B group. It was notable that the protein expression of β-NGF in the undescended testis of the HN group was markedly higher compared with the NC, HC, LN and B groups(Fig. 3A–E).

The positive staining, colored brown, of HoxA10 was mainly localized in the cell nucleus of the germ cells, interstitial cells and leydig cells in the testes. Notably, the protein expression of HoxA10 in the cryptorchidism of the HN group was markedly higher compared with the NC, HC, LN and B groups (Fig. 4A–E).

Compared with the cells of the B group, the NC group exhibited pathological modifications, including reduced nuclear chromatin, vacuolar degeneration in ribosomes, thickened and damaged nuclear membrane, widening of the perinuclear space and nuclear pyknosis. Following treatment with a high dose of NGF, the testes of the HN group appeared to resemble normal testes, with fewer of the pathological changes mentioned above (Fig. 5A–E).